There is much about early development that is not understood. At some point after fertilization the zygotic genome is activated, and genes that are necessary for upcoming developmental events are transcribed. Many early genes have been characterized and placed into gene networks, but how they come to be activated at the proper time, in the proper order, and in a robust manner is unclear. In the last grant period, it was revealed that the Drosophila embryo uses a simple strategy to collectively and selectively activate genes - the transcription factor Zelda (Zld) recognizes and binds a specific sequence motif (CAGGTA), and subsequently integrates gene networks into a system of coherent and incoherent feed-forward loops. In doing so, Zld drives the major reprogramming event that occurs during the maternal-to-zygotic transition (MZT), where the embryo takes over control of development from the mother. This is the first demonstration of such a regulator in any organism, thus, Zld provides a paradigm for the global coordination of gene networks in early development. Zld resembles a special class of transcription factors called pioneer factors, which are the first to engage target genes, providing competence for future activation when developmental signals come along. The goal of this proposal is to reveal the underlying mechanisms by which Zld functions to regulate zygotic genome activation (ZGA) and to provide robust temporal control to the activities of the key developmental regulators. Zld protein is initially at low levels, rises in concentration by one hour, coincident with ZGA, then decreases in the third hour. The regulation of Zld protein dynamics and how Zld times the on-off state of early genes is unclear. The hypothesis that a threshold level of Zld triggers genome activation will be tested by altering Zld concentrations and monitoring target gene activation. Whether Zld itself is regulated will be assessed, with focus on a potential role of the zld 3' UTR. The idea that Zld binding leads to local remodeling of the chromatin landscape, thus increasing accessibility of other factors, leading to increased expressivity of target genes will be tested. Global changes in chromatin states at enhancers and transcription start sites before ZGA when the genome is inactive, after ZGA when the early set of zygotic genes are transcribed, and later when many early genes are turned off and others are activated will be examined. Importantly, comparing chromatin states in wild-type and zld mutants will reveal if Zld mediates epigenetic changes during the MZT, and if certain chromatin marks prepare genes for future transcriptional activation/repression. Genetic, genomic, and biochemical approaches will be used to test these hypotheses. Key reagents include zld mutants, zld rescue transgenes to alter Zld levels, and transcriptional reporter transgenes to assay Zld target- gene responses to changes in Zld binding sites. The experimental approaches will lead to a greater understanding of zygotic genome reprogramming from a quiescent state to a robustly active state in early development.